One-Step Formulation of Targeted Aggregation ... - ACS Publications

One-Step Formulation of Targeted Aggregation-Induced Emission Dots for Image-Guided Photodynamic Therapy of Cholangiocarcinoma Min Li,†,‡,⊥ Yang Gao,†,⊥ Youyong Yuan,§,⊥ Yuzhe Wu,† Zifang Song,† Ben Zhong Tang,‡,∥ Bin Liu,*,§ and Qi Chang Zheng*,† †

Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China ‡ State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China § Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore, 117585 ∥ Department of Chemistry and Division of Biomedical Engineering, Hong Kong University of Science and Technology, Hong Kong 999077, China S Supporting Information *

ABSTRACT: Photodynamic therapy (PDT) is a palliative technique that can improve median survival with minimal invasion for cholangiocarcinoma (CC) patients. An ideal photosensitizer (PS) is critical to guarantee the efficacy of PDT. However, conventional PSs have some obvious drawbacks, such as lack of specificity and easy aggregation in aqueous media that limit their further application in the clinic. We herein fully take advantage of a red emissive aggregationinduced emission (AIE) PS to fabricate integrin ανβ3 targeted organic AIE dots for image-guided PDT via a simple and straightforward onestep strategy. The obtained AIE dots exhibit high specificity to CC as well as excellent antitumor effect both in vitro and in vivo. Different from conventional PSs and previously reported PS-loaded nanostructures, the AIE dots do not suffer from aggregationcaused fluorescence quenching and reduction in reactive oxygen species production when the AIE PS molecules are in an aggregated state. The significant antitumor effect, as well as good biocompatibility and negligible toxicity, makes the AIE dots promising for future translational research in CC diagnosis and therapy. KEYWORDS: cholangiocarcinoma, aggregation-induced emission, targeted fluorescence imaging, photodynamic therapy, targeted therapy inevitable detrimental side effects.8 In addition, they could easily aggregate in aqueous media through π−π stacking due to their hydrophobic and rigid planar structures, resulting in aggregation-caused fluorescence quenching and dramatic reduction in ROS generation.9−11 Consequently, selective and effective PSs are needed to surmount the aforementioned obstacles. Nanoparticles (NPs) for targeted delivery are a good strategy to overcome the first drawback. Various functionalized NPs have been employed for improving the specificity to tumors and have achieved remarkable progress.12−16 However, the

C

holangiocarcinoma (CC) is a devastating malignancy with a high mortality that is difficult to diagnose.1 The vast majority of patients with CC are poor candidates for curative surgery.2 Photodynamic therapy (PDT) is thus a palliative technique that can improve life quality and median survival with minimal invasion for these patients.3−6 PDT utilizes a combination of photosensitizer (PS), light, and oxygen to exert cytotoxicity toward target cells with reactive oxygen species (ROS) produced during the photochemical reactions, which can induce apoptosis or necrosis in treated cells.7 An ideal PS is critical to guarantee the efficacy of PDT. However, conventional PSs have obvious drawbacks. PSs such as porphyrin and its derivatives could also accumulate in normal tissues due to the lack of specificity, which can yield © 2017 American Chemical Society

Received: January 15, 2017 Accepted: April 6, 2017 Published: April 6, 2017 3922

DOI: 10.1021/acsnano.7b00312 ACS Nano 2017, 11, 3922−3932

Article

www.acsnano.org

Article

ACS Nano

Scheme 1. Schematic illustration of T-TTD dots-mediated PDT in a human cholangiocarcinoma xenograft mouse model. The process includes four steps: (1) intravenous injection of T-TTD dots via the tail vein; (2) NP accumulation in tumor tissue via passive targeting, which is well known as the EPR effect; (3) active targeting via ligand−receptor-mediated endocytosis; (4) ROS generation to induce cell apoptosis or necrosis upon light irradiation. TTD: 2-(2,6-bis((E)-4-(phenyl(4′-(1,2,2triphenylvinyl)-[1,1′-biphenyl]-4-yl)amino)styryl)-4H-pyran-4-ylidene)malononitrile; T-TTD: targeted TTD.

put forward for targeted and image-guided PDT of CC. Briefly, upon intravenous injection of the synthesized T-TTD dots into a tumor-bearing mouse, these dots could accumulate into tumor interstitial fluid through fenestration by the enhanced permeability and retention (EPR) effect due to the abnormal vascular architecture. As integrin ανβ3 is overexpressed on the surface of CC cells compared to normal cells, specific interaction between integrin ανβ3 and cRGD on T-TTD dots will trigger the process of ligand−receptor-mediated endocytosis, which plays a critical role in facilitating internalization of the dots into the targeted tumor cells. Upon light illumination at a proper time point, the T-TTD dots in the tumor site will emit red fluorescence to depict the tumor outline. The image will guide further photodynamic therapy to induce tumor cell death. Detailed experiments were conducted to prove the feasibility of our hypothesis and assess the possibility of theranostic AIE dots for potential clinical applications.

second drawback is difficult to address because it is the intrinsic property of conventional PSs. The situation is even more severe when PSs are encapsulated into nanocarriers, which results in significant reduction of both fluorescence and photosensitization.17 Fortunately, fluorogens with aggregation-induced emission characteristics (AIEgens) provide a potential opportunity to overcome this limitation. AIEgens are a special type of molecules that are nonemissive in the molecularly dissolved state but are induced to emit bright fluorescence by aggregation due to the restriction of intramolecular motions.18 These distinctive properties have allowed us to develop specific lightup probes and AIE dots for protein detection, targeted cancer cell and vascular imaging, stem cell tracking, and continuous monitoring of biological processes.19−24 Intriguingly, several AIEgens were also designed to generate ROS upon light irradiation.25−27 Different from traditional PSs, which suffer from quenched fluorescence and reduced ROS production in the aggregated state, AIE PSs offer bright fluorescence with efficient ROS production in NP format.28 More recently, AIE PSs have been integrated into both theranostic probes and organic dots for image-guided cancer cell ablation,10,29 lightcontrolled drug release, and photoactivated gene delivery.30,31 Due to the relatively short absorption wavelengths for most of the AIE PSs available, all these studies are limited to in vitro analysis. In this contribution, we take advantage of a red emissive AIE PS (TTD) with long absorption wavelength to develop a targeted theranostic AIE dot (T-TTD dot) for image-guided PDT using a CC model. A simple and straightforward strategy is also developed to realize one-step formulation of targeted theranostic AIE dots. Scheme 1 represents the hypothesis we

RESULTS AND DISCUSSION The AIE molecule of 2-(2,6-bis((E)-4-(phenyl(4′-(1,2,2triphenylvinyl)-[1,1′-biphenyl]-4-yl)amino)styryl)-4H-pyran-4ylidene)malononitrile (TTD) was synthesized according to our previous report.32 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPEPEG2000-Mal) was conjugated with thiolated cRGD (cRGDSH) through click reaction between maleimide and −SH to yield the DSPE-PEG2000-cRGD (Figures S1 and S2). The TTTD dots were prepared by a modified nanoprecipitation method using DSPE-mPEG and DSPE-PEG2000-cRGD to form NPs with TTD molecules encapsulated as the hydrophobic 3923


Article

ACS Nano

Figure 1. Synthesis of T-TTD dots and characterization. (a) Schematic illustration of the T-TTD dots formation. (b) Size distribution and TEM image (inset) of T-TTD dots. (c) UV−vis absorption and emission (λex = 502 nm) spectra of T-TTD dots. (d) UV−vis absorption spectra changes of the ROS indicator ABDA mixed with T-TTD dots for different time durations of light irradiation (250 mW/cm2).

Figure 2. Expression of integrin αvβ3 in specimen and in human primary and tumor-derived cell lines. (a and b) Relative expression of integrin αν and β3 genes in HK-2, L-O2, and QBC939 cells. All data are expressed as mean ± SD of three separate experiments. *p < 0.05, **p < 0.01. (c) Immunofluorescence (IF) images stained for integrin ανβ3 (green) and nuclei (blue) in QBC939, L-O2, and HK-2 cells. (d) Typical H&E stain and integrin ανβ3 expression of tumor and peripheral nontumor tissues. Scale bars: 50 μm.

3924


Article

ACS Nano

Figure 3. Targeted imaging of T-TTD dots and receptor blocking experiments in vitro. (a) Fluorescent images of QBC939, L-O2, and HK-2 cells after incubation with T-TTD dots (5 μg/mL) for 4 h. Red fluorescence is from the T-TTD dots. Blue fluorescence labels the nuclei, and green fluorescence labels the cytoskeleton. Scale bars: 50 μm. (b) Quantitative analysis of fluorescence intensity of QBC939, L-O2, and HK-2 cells after incubation with T-TTD dots (5 μg/mL) for 4 h. (c) Fluorescent images of QBC939 after incubation with T-TTD dots (5 μg/mL) for 4 h without or with pretreatment with cilengitide. Scale bars: 50 μm. (d) Relative fluorescence intensity of QBC939 after incubation with T-TTD dots without or with receptor blocking.

addition, an about 10-fold increase of the β3 subunit mRNA, which plays a decisive role in integrin ανβ3 function, was detected in QBC939 cells compared to the normal cell lines (Figure 2b). Immunofluorescence corresponding to the gene levels of QBC939 cells showed strong integrin α ν β 3 fluorescence signal, whereas the signal is low for both L-O2 and HK-2 cells (Figure 2c). Furthermore, the integrin ανβ3 expression was tested in 20 pairs of human CC and peripheral nontumor tissues. The expression of ανβ3 was positive in 80% of CC patients (16/20), whereas the peritumor tissues were consistently negative (Figure 2d). These results suggest that the overexpressing integrin ανβ3 on CC cells could provide abundant binding sites for cRGD peptide for ligand− receptor-mediated endocytosis. To verify the targeted imaging of T-TTD dots to CC, fluorescence imaging was performed in vitro and in vivo. The application of the T-TTD dots in targeted cellular fluorescence imaging was studied with confocal laser scanning microscopy (CLSM). After incubation with T-TTD dots (5 μg/mL), QBC939, L-O2, and HK-2 cells were imaged by CLSM upon excitation at 543 nm with a collection of fluorescent signals above 650 nm. As shown in Figure 3a, a robust red fluorescent signal was observed in QBC939 cells, which is localized in the cytoplasm around the nuclei. In contrast, weak fluorescence was observed in the L-O2 and HK-2 cells. The uptake of T-TTD dots by QBC939, L-O2, and HK-2 cells was also quantitatively evaluated by flow cytometry. Histograms shown in Figure 3b indicate that the average fluorescence intensity of QBC939 cells incubated with T-TTD dots is about 6-fold higher than normal cells, while the fluorescent baseline of three types of cells is at the same level (Figure S4). Receptor blocking experiments were also carried out to investigate the uptake mechanism of TTTD dots. In these experiments, integrin ανβ3 inhibitor (cilengitide) was first incubated with QBC939 cells before TTTD dots were added. As shown in Figure 3c,d, the red fluorescent signal of T-TTD dots is dramatically reduced to

core (Figure 1a) and cRGD exposed to the aqueous media. The dynamic light scattering (DLS) and transmission electron microscope (TEM) study of T-TTD dots show an average size of ∼40 nm (Figure 1b). The size change of the dots at different days in a week was measured by DLS, which reveals that the TTTD dots are stable at room temperature (Figure S3). The absorption and emission spectra of T-TTD dots are shown in Figure 1c, which have an absorption peak centered at 502 nm with an emission maximum at 660 nm. The ROS generation of T-TTD dots was studied by measuring the absorbance decrease of 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA) upon white light irradiation. As shown in Figure 1d, the absorption of ABDA decreases gradually upon light irradiation, as the anthracene moiety in ABDA can efficiently react with ROS. The absorbance of ABDA at 400 nm is decreased to 28% of its original value after 15 min of light irradiation, confirming the ROS generation of T-TTD dots. The ROS quantum yield (Φ) of T-TTD dots was determined to be 0.51 using Rose Bengal (RB) as the standard PS (ΦRB = 0.75 in water), which is comparable to clinically used PSs such as Photofrin (Φ = 0.28) or Laserphyrin (Φ = 0.48).33 Specific receptor expression on the cell membrane is a prerequisite of ligand−receptor-mediated endocytosis. Integrin ανβ3 is strongly up-regulated in many solid tumor cells and endothelial cells of new vasculature in tumor tissues, while it is deficient in most normal organs, suggesting that it is a specific and potential binding target of interest.34 However, the expression of integrin αvβ3 in CC is rarely reported. As a consequence, we first evaluated the expression of integrin αvβ3 in specimens from patients with CC and in human primary and tumor-derived cell lines by immunohisto- and cyto-chemistry, as well as by RT-qPCR. A significant up-regulation of αν and β3 mRNA was detected in QBC939 cells compared to normal LO2 and HK-2 cells. For example, integrin αν mRNA expression is about 2-fold and 4-fold higher in QBC939 cells as compared to that in L-O2 and HK-2 cells, respectively (Figure 2a). In 3925


Article

ACS Nano

Figure 4. Targeted imaging of T-TTD dots and receptor blocking experiments in vivo. (a) Biodistribution of T-TTD dots in tumor-bearing mice after intravenous injection of T-TTD dots (30 mg/kg) at different times. White circles indicate the tumor. (b) Ex vivo fluorescence imaging of various organs and tumor tissue from mice injected with T-TTD dots. The mice were sacrificed at 12 h postinjection. (c) Biodistribution of T-TTD dots in tumor-bearing mice 8 h after intravenous injection of T-TTD dots (30 mg/kg) without or with blocking the receptors. White circles indicate the tumor. (d) Quantitative analysis of organ distribution of T-TTD dots in tumor-bearing mice 8 h after intravenous injection with or without blocking the receptors with cilengitide. Data represent mean ± SD (n = 3). **p < 0.01. (e) Blood circulation curves of T-TTD dots in the blood at different time points postinjection. The y-axis shows the percentage of injected dose per gram of tissue. Error bars are based on 3 mice per group.

accumulated in the liver and intestine, the greatest amounts of NPs appear in tumor tissue. To confirm the receptor specificity, blocking experiments were also performed in vivo. Cilengitide was injected 30 min before the administration of T-TTD dots. As shown in Figure 4c, the tumor uptake of T-TTD dots at 8 h postinjection is significantly inhibited by the preinjection of cilengitide. Figures S5 shows the ex vivo fluorescence imaging of various organs from tumor-bearing mice intravenously injected with T-TTD dots with or without blocking the receptors with cilengitide at 8 h postinjection. Brighter fluorescent signal is observed in tumor tissues from the mice without blocking. The average fluorescence intensity of each harvested organ at 8 h postinjection was measured for a semiquantitative biodistribution analysis, which is shown in Figure 4d. The biodistribution of T-TTD dots in normal tissues is similar for mice groups with and without blocking. Noteworthy is that the average fluorescence intensity in tumor tissues from the mice without blocking is ∼2.7-fold higher than that from the cilengitidepretreated mice. In addition, the difference in fluorescence intensity at the tumor site between the two groups (n = 3 mice

about 20% of that in cells without incubation of cilengitide. These results demonstrate that the T-TTD dots were taken up through integrin ανβ3 receptor-mediated endocytosis. Next, the uptake of T-TTD dots by tumor tissue in vivo was investigated by a noninvasive fluorescence imaging system. QBC939 tumor-bearing mice were injected with T-TTD dots (125 μL, 5 mg/mL) via the tail vein, and fluorescent images were subsequently recorded at different time intervals. As shown in Figure 4a, the NPs reveal a time-dependent biodistribution and tumor preferential profile in the mice. Although the dots are widely dispersed among the whole body within 2 h postinjection, they have a tendency to accumulate in the tumor tissue as the time elapses. A significant increase of fluorescence is observed in tumor 8 h postinjection. The fluorescent signals gradually decrease 24 h postinjection through the process of circulation. To further verify the biodistribution of these NPs under systemic circulation, the mice after injection of T-TTD dots for 12 h were sacrificed, and various organs and tissues were isolated to image ex vivo. As shown in Figure 4b, although plenty of T-TTD dots are 3926


Article

ACS Nano

Figure 5. Therapeutic effect of T-TTD dots-mediated PDT in vitro and in vivo. (a) Detection of intracellular ROS production by DCF-DA in QBC939, L-O2, and HK-2 cells after incubation with T-TTD dots (5 μg/mL) followed by irradiation. Scale bar: 50 μm. (b) Inhibition of viability of QBC939, L-O2, and HK-2 cells pretreated with different concentrations of T-TTD dots followed by 3 min of irradiation. (c) Viability of cells pretreated with the same concentration of T-TTD dots (5 μg/mL) and then subjected to different irradiation times. Data represent mean ± SD, n = 3. (d) Antitumor effect of different treatments in vivo. Photographs of xenografted tumor (white circle) 3 days after different treatments (top row). H&E staining for pathological changes in tumor sections (middle row). TUNEL staining (green) for apoptosis in tumor sections (bottom row). Blue fluorescence localizes the nuclei of cells. Scale bar: 100 μm. Laser parameters (wavelength, 532 nm; laser power, 250 mW/cm2). (e) The volume growth curves of tumors at different time points post-treatment in 4 groups (n = 4 per group). Data represent mean ± SD; **p < 0.01. (f) Survival curves of tumor-bearing mice in 4 groups with different treatments (n = 7 per group). *p < 0.05.

per group) is statistically significant (p < 0.01). Together, these results indicate that the dots with surface cRGD functionaliza-

tion are able to greatly accumulate in tumor tissues not only through passive targeting resulting from the EPR effect but also 3927


Article

ACS Nano

Figure 6. Hemocompatibility evaluation of T-TTD dots. (a) Photographs of hemolysis assay after incubation with distilled water (positive control), saline (negative control), and T-TTD dots for 1, 2, and 3 h, respectively. The presence of large amounts of hemoglobin in the supernatant is observed only in the positive control tube. (b) Optical microscopic observation of the dispersion states of the erythrocytes after incubation with distilled water, saline, and T-TTD dots for 1, 2, and 3 h. Scale bars: 20 μm. (c) Hemolysis rate of each group. Data represent mean ± SD (n = 3).

via ligand-based active targeting effect. In order to study the circulation profile, T-TTD dots were intravenously injected into BALB/c mice. Blood was drawn from the tail vein at different time points after injection. The dot concentration was determined by measuring the fluorescence intensity of T-TTD dots after subtracting the blank blood sample from an untreated control mouse as shown in Figure 4e. The in vivo blood circulation half-life of T-TTD dots was calculated to be ∼6 h, which guaranteed them to have enough time to gradually accumulate at the tumor site. Cytotoxic reactive oxygen species, especially singlet oxygen (1O2), have been proved to result in irreversible cell damage and cause cell death and tumor destruction in PDT.35 To evaluate the ROS productivity by T-TTD dots after cellular uptake, a cell-permeable fluorescent dye, dichlorofluorescein diacetate (DCF-DA), was chosen as the indicator. After 4 h of incubation of T-TTD dots, large quantities of ROS were generated in QBC939 cells upon light irradiation, while no obvious ROS was detected in cells without incubation or irradiation (Figure S6). Moreover, intense green fluorescence was observed inside the QBC939 cells after irradiation, while negligible fluorescence was observed in L-O2 and HK-2 cells (Figure 5a), demonstrating efficient ROS generation from the T-TTD dots in QBC939 cells. It should be noted that the cell viabilities are very high for T-TTD dots-incubated cells without light irradiation or for cells upon light irradiation without TTTD dots incubation, which indicates low cytotoxicity of the dots on QBC939, L-O2, and HK-2 cells (Figure S7). To further study the cytotoxicity of ROS to viable cells, FITC-annexin V was used to distinguish dead cells from viable ones. After incubating QBC939 cells with T-TTD dots followed by irradiation, green fluorescence was clearly observed from the cell membrane, indicating that the cells underwent cell apoptosis. No fluorescent signal was observed in L-O2 and HK-2 cells, which indicated negligible phototoxicity of the NPs on the cells due to their poor uptake (Figure S8). Additionally, to better illustrate the effectiveness of photodynamic cell ablation on QBC939 cells, calcein-AM/propidium iodide (PI) dual fluorescent labeling was performed to differentiate live and dead cells. As shown in Figure S9, the T-TTD dots and light alone have negligible cytotoxity on QBC939 cells. However, cells treated with T-TTD dots followed by light irradiation show increased PI stain (red fluorescence) indicating cells death.

To understand the relationship between phototoxicity, dot concentration, and irradiation time, cell viability was examined 24 h after PDT. Concentration- and time-dependent cell death induced by PDT was observed in QBC939 cells (Figure 5b,c). The ROS productivity was also tested using DCF-DA as the indicator, and it was found that the fluorescence intensity enhanced with the increase of T-TTD dots concentration (Figure S10a). In addition, the morphological changes of cells were observed 6 h after PDT. As shown in Figure S10b, no morphological changes were observed in control cells upon irradiation without T-TTD dots treatment. Incidence of cell shrinkage and swelling, defining hallmarks of apoptosis and necrosis, respectively, was significantly increased with the elevation of T-TTD dot concentration. The same phenomenon was also observed in cells upon incubation with 5 μg/mL of TTTD dots, followed by irradiation at various periods of time (Figure S10c,d). In addition, the proportion of necrosis was scaled up with the increase of T-TTD dot concentration and irradiation time (Figure S10b,d), suggesting that the T-TTD dot-mediated PDT has induced both apoptotic and necrotic cell death. We further noticed that the increase in both concentration and irradiation time could change the dominant cell death process from apoptosis to necrosis. These data indicate that the PDT mediated by T-TTD dots exhibits a concentration- and time-dependent profile, which facilitates the control of therapeutic potency during PDT. Encouraged by the promising results of targeted tumor imaging and antitumor efficacy in vitro, we further evaluated the therapeutic effect of T-TTD dots-mediated PDT in vivo. Tumor-bearing mice with a similar tumor size of 60 mm3 were divided into four groups. Group 1 (negative control) was only injected with saline via the tail vein. Group 2 was injected with saline followed by laser irradiation (530 nm, 250 mW/cm2) 12 h after injection. Group 3 was injected with T-TTD dots without laser irradiation, while group 4 was injected with dots followed by laser irradiation (530 nm, 250 mW/cm2). As shown in Figure 5d, obvious tumor shrinkage was observed 3 days after PDT in group 4. In contrast, no noticeable changes were observed in the other three groups. Moreover, the tumor sizes were also measured for 16 days after different treatments. The tumor sizes were reduced significantly only in group 4 (p < 0.01), whereas the tumors in the other groups revealed fast growth (Figure 5e). The histological tumor tissue analysis also revealed the efficacy of PDT treatment. After 3 days of different 3928


Article

ACS Nano

Figure 7. Toxicity evaluation of T-TTD dots in vivo. (a) Body weight changes of Balb/c mice after intravenous injection of saline (negative control) and T-TTD dots, respectively. Data represent mean ± SD (n = 4). (b) Blood biochemical data of the two groups. (c). Images of various H&E-stained organ slices from mice 7 days after different treatments. Scale bar: 50 μm.

blood for biochemical examination, and various organs were isolated for histological analyses. Indexes reflecting hepatic function including albumin (ALB) and alanine transaminase (ALT), as well as renal-related indexes including blood urea nitrogen (BUN) and serum creatinine (Cr), were tested. No physiologically significant difference (p > 0.05) was observed between the two groups, as shown in Figure 7b, indicating that the administration of T-TTD dots caused no obvious dysfunction to the liver and kidney. Furthermore, H&E staining of various organs revealed no pathological change after dot injection (Figure 7c, Figure S11). These results demonstrate that T-TTD dots are safe biomaterials for in vivo applications.

treatments, tumor tissues from each group were isolated for H&E and TUNEL staining. As shown in Figure 5d, obvious apoptosis and necrosis suggest that more significant damage is observed in the group 4 tumors than in other groups. In addition, the mice in the PDT group have a significant extension of survival (p < 0.05), as shown in Figure 5f. Together, these results suggest that T-TTD dots-mediated PDT is a desirable therapeutic strategy for CC ablation. Hemocompatibility is a critical parameter to evaluate biomaterials for in vivo biomedical applications.36 A hemolysis test was used to assess the compatibility of T-TTD dots with erythrocytes. As shown in Figure 6a, no distinct hemolysis could be observed in saline (negative control) and the T-TTD dot groups under the studied concentration range, whereas obvious hemolysis occurred in distilled water (positive control). Similarly, no morphological change of erythrocytes was observed in the dot groups, while erythrocytolysis occurred in the positive control (Figure 6b). Hemoglobin release after exposure to various concentrations of T-TTD dots was measured by spectrophotometer. Quantitative analyses showed that the hemolysis rates of the NPs, even at 666.8 μg/mL (Figure 6c), were far less than the critical safe hemolytic ratio set by ISO/TR7406.37 These results demonstrate that the TTTD dots exhibit excellent hemocompatibility over the studied concentration range, allowing for their safe systemic administration. Low toxicity or even nontoxicity is the prerequisite for biomaterials to be used in the human body.38 In this regard, 250 μL of T-TTD dots (5 mg/mL), which was double the dose of that used for imaging and PDT, was intravenously injected into healthy mice to examine its toxicity. After administration, body weight changes of mice in the dot group and saline group (negative control) were monitored for 7 days. As shown in Figure 7a, no noticeable body weight losses were observed in either group. The mice were subsequently sacrificed to collect

CONCLUSIONS In summary, we report smart organic dots based on a red emissive AIE PS for image-guided PDT. These one-step formulated and targeted theranostic dots can effectively stain CC with good biocompatibility and negligible toxicity. Compared with conventional PSs and previously reported PSloaded nanostructures,39,40 the AIE PS does not involve any aggregation-caused quenching effect; instead, it reveals high brightness and good ROS productivity even in an aggregated state. The significant antitumor effect upon light irradiation in a dose- and time-dependent manner makes the AIE dots promising for future translational research in CC diagnosis and therapy. METHODS Conjugation of cRGD with DSPE-PEG2000-Mal. Thiolated cRGD was conjugated to DSPE-PEG2000-Mal by the following procedure. DSPE-PEG2000-Mal (5 mg) was dissolved in DMSO (0.5 mL) followed by the addition of excess thiolated cRGD (3 equiv) and stirred at room temperature for 6 h. Then the reaction mixture was poured into Milli-Q water (10 mL) and further washed with water (3 3929


Article

ACS Nano

and incubated at 37 °C. Following incubation with T-TTD dots for 4 h, the cells were washed twice with PBS and then exposed to light irradiation for different times at a power density of 250 mW/cm2. The cells were further incubated for 24 h. The viability tests for cells upon incubation with T-TTD dots without light irradiation and cells with light irradiation but without T-TTD dots incubation were also performed. After different treatments, the cells were further incubated for 24 h at 37 °C. This was followed by the addition of 10 μL of CCK8 solution into each well and then incubated at 37 °C for 1 h. The absorbance was measured at 450 nm by using a Multiskan GO microplate spectrophotometer (Thermo Scientific, USA). Cell viability was expressed by the ratio of the absorbance of CCK-8 in cells incubated with dots to that of the cells incubated with culture medium only. Animal Model. Nude mice weighing 20 g were purchased from HFK Bioscience Co. (Beijing, China). All of the animal studies were performed in compliance with guidelines set by the Animal Care Committee at Tongji Medical College. To establish the QBC939 tumor model, 1 × 106 cells were inoculated subcutaneously into the right front flanks of female BALB/c nude mice. Tumor growth was measured using a caliper, and the tumor volume was calculated using the following formula: volume = ((tumor length) × (tumor width)2)/ 2. In Vivo Imaging of T-TTD Dots in QBC939-Xenografted Nude Mice. The tumor-bearing mice models were established as described above. When tumor volume reached a mean size of about 100 mm3, T-TTD dots (125 μL, with TTD loading of 0.92 wt %) were injected via tail vein at a concentration of 5 mg/mL based on TTD. After administration, in vivo real-time fluorescence imaging was performed with an In-Vivo FX PRO (Bruker, Germany). Scans were carried out at 2, 8, 24, and 48 h postinjection. Moreover, the nude mice were sacrificed at 12 h after administration, and the fluorescence intensity of various organs was measured. Light with a central wavelength of 530 nm was selected as the excitation wavelength. In vivo spectral imaging from 600 to 900 nm (with 100 nm steps) was collected. Exposure time for each image frame was 1 s. For the blocking experiment in vivo, tumor-bearing mice were intravenously injected with 30 mg/kg T-TTD dots, and the blocking mice were injected with cilengitide (100 μg) 30 min before the administration of T-TTD dots. Mice were subjected to optical imaging using the same protocol. The quantitative analysis of organ distribution of T-TTD dots in tumor-bearing mice 8 h after intravenous injection of T-TTD dots (30 mg/kg) with or without blocking the receptors was also performed. The nude mice were sacrificed at 8 h after intravenous injection, and the fluorescence intensity of various organs and tumors of both groups (n = 3 in each group) was measured. In Vivo PDT Efficacy of T-TTD Dots. QBC939 cell tumor-bearing BALB/c nude mice with a uniform tumor size were chosen for in vivo PDT studies. When the tumors reached a volume of about 60 mm3, the mice were randomly divided into four groups (n = 15 in each group). The four groups were treated as follows: Group 1 (negative control) was only injected with saline via tail vein; group 2 was injected with saline followed by laser irradiation (530 nm, 153 J/cm2, 250 mW/cm2) 12 h after injection; group 3 was injected with T-TTD dots without laser irradiation; group 4 was injected with T-TTD dots followed by irradiation. Three days after different treatments, 4 mice from each group were sacrificed, and the tumors were excised. Tumor tissues were fixed with 10% neutral buffered formalin and embedded in paraffin. Slices were stained with hematoxylin-eosin (H&E) and assessed by TUNEL assay to confirm the PDT efficacy of T-TTD dots. Tumor sizes were also measured for 16 days after different treatments (n = 4). The rest of the mice in each group (n = 7) were treated with the same method every week, and survival time was recorded. Detection of Cell Apoptosis by the TUNEL Assay. Apoptosis and necrosis levels of tumor tissues after different treatment were further assessed by a one-step TUNEL apoptosis assay kit. The experimental procedures were performed according to the manufacturer’s protocol. Briefly, paraffin-embedded sections of tumor tissues were deparaffinized and treated with 20 μg/mL of proteinase K at 37 °C for 10 min. After rinsing, sections were incubated with TUNEL

mL × 5 times) by ultrafiltration (3000 MWCO, Amicon, Millipore Corporation, Bedford, MA, USA). DSPE-PEG2000-cRGD was obtained as a white powder after freeze-drying (4.66 mg, 76% yield). ESI-MS: [M + H]+ m/z = 3416.75. Preparation of Targeted T-TTD Dots. A THF solution (1 mL) containing DSPE-mPEG2000 (1.0 mg), DSPE-PEG2000-cRGD (0.1 mg), and TTD (0.5 mg) was poured into water (10 mL). This was followed by sonicating the mixture for 2 min at 12 W output using a microtip probe sonicator (XL2000, Misonix Incorporated, NY, USA). The mixture was then stirred at room temperature overnight to evaporate the organic solvent. The NP suspension was further filtered with a 0.2 μm syringe filter to obtain T-TTD dots. The amount of TTD encapsulated in each dot was calculated by measuring the absorbance at 500 nm and comparing with a standard curve. Encapsulation efficiency (%) was calculated as follows: encapsulation efficiency = [amount of TTD in the dots]/[total amount of TTD used] × 100. Detection of ROS in Solution. The ROS generation was studied by using ABDA as an indicator as the absorbance of ABDA decreases upon reaction with ROS. For ROS detection, the stock solution of ABDA in DMSO was mixed with T-TTD dots (1 μg/mL) and exposed to white light irradiation (250 mW/cm2). The decomposition of ABDA was monitored by the absorbance decrease. Cell Culture. The human cholangiocarcinoma QBC939 cells and human normal liver L-O2 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China), and human kidney proximal tubular (HK-2) cells were purchased from American Type Culture Collection. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. In Vitro Cell Fluorescence Imaging. Exponentially growing QBC939, L-O2, and HK-2 cells were seeded in slide chambers. After 80% confluence, the culture medium was removed and washed twice with phosphate-buffered saline (PBS). Following incubation with TTTD dots (5 μg/mL) for 4 h, cells were washed three times with PBS and fixed with 4% paraformaldehyde for 10 min. After permeabilized with 0.1% Triton X-100 in PBS for 6 min, cells were washed with PBS three times and stained with 10 μg/mL of FITC-Phalloidin at 37 °C in the dark for 1 h. Nuclei were stained with Hoechst 33342 (1:1000) for 5 min. Between each step, cells were washed three times. Cell images were acquired using a laser confocal microscope (Nikon, Japan). The excitation was 543 nm, and the emission was collected above 650 nm. For the receptor blocking experiments, cilengitide was used as the integrin ανβ3 inhibitor. Cilengitide (0.1 μg/mL) was first incubated with QBC939 cells for 6 h before T-TTD dots were added, and fluorescent images were acquired using the same protocol as described above. Quantitative Analysis of Cellular Uptake by Flow Cytometry. QBC939, L-O2, and HK-2 cells were seeded respectively onto a sixwell plate at a number of 3 × 105 per well and incubated overnight before further experiments. After cells attached, the culture medium was removed and cells were incubated with T-TTD dots (5 μg/mL). After incubation for 4 h, the medium was discarded and the cells were washed three times with PBS. Cells were harvested, and the cellular uptake efficiency was determined by flow cytometry. Intracellular ROS Detection. ROS production inside cells after light irradiation was detected using the fluorescent probe DCFH-DA according to the manufacturer’s instructions. QBC939, L-O2, and HK2 cells were seeded respectively onto a 12-well plate at a number of 1 × 105. After the cells were attached, the culture medium was removed and washed twice with PBS. After incubation with T-TTD dots for 4 h, DCFH-DA was loaded into cells. After 20 min of incubation, cells were washed twice with PBS and then exposed to light irradiation at the power density of 250 mW/cm2. Fluorescence intensity was observed by a fluorescence microscope (IX71, Olympus, Japan) after irradiation. Measurement of Cell Viability. Cytotoxicity of the T-TTD dots against different cells was measured using a CCK8, a commercially available assay kit. Briefly, QBC939, L-O2, and HK-2 cells were seeded in 96-well plates at 1 × 104 cells per well. After cell adherence, the medium was replaced with the T-TTD dots at different concentrations 3930


Article

ACS Nano reaction mixture for 1 h at 37 °C. Finally, the sections were stained with Hoechst 33342 for 15 min to localize nuclei of cells. Apoptotic cells in each experimental group were imaged by fluorescence microscopy. Hemolysis Assay. Fresh blood was obtained by heart puncture from BALB/c mice and diluted using physiological saline. Red blood cells (RBCs) were then isolated from the serum by centrifugation at 3000 rpm for 10 min. After careful washing three times, a suspension of RBCs was added into the T-TTD dots solution at systematically varied concentrations and mixed completely. The mixture was incubated in a 5% CO2 atmosphere at 37 °C for 1, 2, and 3 h, respectively. After incubation, all suspensions were centrifuged at 3000 rpm for 5 min, and then photos were taken for each sample. The supernatant of each tube was transferred to a 96-well plate. The OD values of supernatants were measured with a Multiskan GO microplate spectrophotometer (Thermo Scientific, USA) at 577 nm. The precipitation of each tube was used to make the cell smear to observe the morphological changes of erythrocyte. The hemolysis ratio of RBCs was calculated using the following formula: Hemolysis (%) = (ODsample − ODnegative control)/(ODpositive control − ODnegative control) × 100. Physiological saline and distilled water were used as the negative and positive controls, respectively. In Vivo Toxicity Assay. In order to evaluate the toxicity and side effects of T-TTD dots, healthy and tumor-free BALB/c mice were used as subjects. The mice were randomly assigned to 2 groups, and each group contained 4 mice. On the zeroth day, one group of mice were injected with 250 μL of saline via tail vein, while the other groups were injected with the same dose of T-TTD dots at a concentration of 5 mg/mL. After injection, weights and clinical situations of all the mice were scrutinized every day for the following 7 days. On the seventh day, all the mice were anesthetized and blood samples were collected through cardiac puncture for further detection. The blood samples were then centrifuged at 3000 rpm for 20 min to obtain the serum. The ALB, ALT, Cr, and BUN levels that could respectively reflect damage to the liver and kidney were tested by Vet Test 8008 (IDEXX, USA). Moreover, all the mice were sacrificed, and the normal organs of mice including the liver, spleen, kidney, and heart were excised for histology observations. The organs were fixed in 10% neutral buffered formalin and embedded routinely with paraffin for H&E staining.

for the Central Universities, China (No. 2014QN064). M.L. thanks the support from the Research Grant of Union Hospital, Tongji Medical College, HUST (No. 02.03.2015-74). B.L. is grateful to the Singapore National Research Foundation (R279000-483-281, R279-000-444-281) and National University of Singapore (R279-000-482-133) for financial support.

REFERENCES (1) Khan, S. A.; Thomas, H. C.; Davidson, B. R.; Taylor-Robinson, S. D. Cholangiocarcinoma. Lancet 2005, 366, 1303−1314. (2) Sirica, A. E. Cholangiocarcinoma: Molecular Targeting Strategies for Chemoprevention and Therapy. Hepatology 2005, 41, 5−15. (3) Cosgrove, N. D.; Al-Osaimi, A. M.; Sanoff, H. K.; Morris, M. M.; Read, P. W.; Cox, D. G.; Mann, J. A.; Argo, C. K.; Berg, C. L.; Pelletier, S. J.; Maluf, D. G.; Wang, A. Y. Photodynamic Therapy Provides Local Control of Cholangiocarcinoma in Patients Awaiting Liver Transplantation. Am. J. Transplant. 2014, 14, 466−471. (4) Ortner, M. A.; Liebetruth, J.; Schreiber, S.; Hanft, M.; Wruck, U.; Fusco, V.; Muller, J. M.; Hortnagl, H.; Lochs, H. Photodynamic Therapy of Nonresectable Cholangiocarcinoma. Gastroenterology 1998, 114, 536−542. (5) Ortner, M. E.; Caca, K.; Berr, F.; Liebetruth, J.; Mansmann, U.; Huster, D.; Voderholzer, W.; Schachschal, G.; Mossner, J.; Lochs, H. Successful Photodynamic Therapy for Nonresectable Cholangiocarcinoma: a Randomized Prospective Study. Gastroenterology 2003, 125, 1355−1363. (6) Park, D. H.; Lee, S. S.; Park, S. E.; Lee, J. L.; Choi, J. H.; Choi, H. J.; Jang, J. W.; Kim, H. J.; Eum, J. B.; Seo, D. W.; Lee, S. K.; Kim, M. H.; Lee, J. B. Randomised Phase II Trial of Photodynamic Therapy Plus Oral Fluoropyrimidine, S-1, versus Photodynamic Therapy Alone for Unresectable Hilar Cholangiocarcinoma. Eur. J. Cancer 2014, 50, 1259−1268. (7) Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Gollnick, S. O.; Hahn, S. M.; Hamblin, M. R.; Juzeniene, A.; Kessel, D.; Korbelik, M.; Moan, J.; Mroz, P.; Nowis, D.; Piette, J.; Wilson, B. C.; Golab, J. Photodynamic Therapy of Cancer: an Update. Ca-Cancer J. Clin. 2011, 61, 250−281. (8) Ethirajan, M.; Chen, Y.; Joshi, P.; Pandey, R. K. The Role of Porphyrin Chemistry in Tumor Imaging and Photodynamic Therapy. Chem. Soc. Rev. 2011, 40, 340−362. (9) Sekkat, N.; van den Bergh, H.; Nyokong, T.; Lange, N. Like a Bolt from the Blue: Phthalocyanines in Biomedical Optics. Molecules 2012, 17, 98−144. (10) Yuan, Y.; Zhang, C. J.; Gao, M.; Zhang, R.; Tang, B. Z.; Liu, B. Specific Light-Up Bioprobe with Aggregation-Induced Emission and Activatable Photoactivity for the Targeted and Image-Guided Photodynamic Ablation of Cancer Cells. Angew. Chem., Int. Ed. 2015, 54, 1780−1786. (11) Yuan, Y.; Zhang, C. J.; Liu, B. A Platinum Prodrug Conjugated with A Photosensitizer with Aggregation-Induced Emission (AIE) Characteristics for Drug Activation Monitoring and Combinatorial Photodynamic-Chemotherapy Against Cisplatin Resistant Cancer Cells. Chem. Commun. (Cambridge, U. K.) 2015, 51, 8626−8629. (12) Nguyen, K. T.; Zhao, Y. Engineered Hybrid Nanoparticles for On-Demand Diagnostics and Therapeutics. Acc. Chem. Res. 2015, 48, 3016−3025. (13) Wang, X.; Wang, X.; Guo, Z. Functionalization of Platinum Complexes for Biomedical Applications. Acc. Chem. Res. 2015, 48, 2622−2631. (14) Barreto, J. A.; O’Malley, W.; Kubeil, M.; Graham, B.; Stephan, H.; Spiccia, L. Nanomaterials: Applications in Cancer Imaging and Therapy. Adv. Mater. 2011, 23, H18−40. (15) Li, M.; Zhang, W.; Wang, B.; Gao, Y.; Song, Z.; Zheng, Q. C. Ligand-Based Targeted Therapy: a Novel Strategy for Hepatocellular Carcinoma. Int. J. Nanomed. 2016, 11, 5645−5669. (16) Sun, Y.; Hu, H.; Zhao, N.; Xia, T.; Yu, B.; Shen, C.; Xu, F. J. Multifunctional Polycationic Photosensitizer Conjugates with Rich

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00312. Additional information and figures (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail (B. Liu): [email protected]. *E-mail (Q. C. Zheng): [email protected]. ORCID

Bin Liu: 0000-0002-0956-2777 Qi Chang Zheng: 0000-0003-0917-8545 Author Contributions ⊥

M. Li, Y. Gao, and Y. Yuan contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Open Fund of the State Key Laboratory of Luminescent Materials and Devices (South China University of Technology, No. 2017-skllmd-02), the National Science Foundation of China (No. 81372668, No. 81502527), Natural Science Foundation of Hubei Province, China (No. 2015CFB527), and Fundamental Research Funds 3931


Article

ACS Nano Hydroxyl Groups for Versatile Water-Soluble Photodynamic Therapy Nanoplatforms. Biomaterials 2017, 117, 77−91. (17) Moan, J.; Berg, K.; Iani, V. Action Spectra of Dyes Relevant for Photodynamic Therapy. In Photodynamic Tumor Therapy 2nd and 3rd Generation Photosensitizers; Mose, J. G., Ed.; Harwood Academic Publishers: Amsterdam, 1998; pp 1169−1181. (18) Mei, J.; Hong, Y.; Lam, J. W.; Qin, A.; Tang, Y.; Tang, B. Z. Aggregation-Induced Emission: the Whole Is More Brilliant than the Parts. Adv. Mater. 2014, 26, 5429−5479. (19) Ding, D.; Li, K.; Liu, B.; Tang, B. Z. Bioprobes Based on AIE Fluorogens. Acc. Chem. Res. 2013, 46, 2441−2453. (20) Li, K.; Liu, B. Polymer-Encapsulated Organic Nanoparticles for Fluorescence and Photoacoustic Imaging. Chem. Soc. Rev. 2014, 43, 6570−6597. (21) Liang, J.; Tang, B. Z.; Liu, B. Specific Light-Up Bioprobes Based on AIEgen Conjugates. Chem. Soc. Rev. 2015, 44, 2798−2811. (22) Feng, G.; Liu, B. Multifunctional AIEgens for Future Theranostics. Small 2016, 12, 6528−6535. (23) Ding, D.; Mao, D.; Li, K.; Wang, X.; Qin, W.; Liu, R.; Chiam, D. S.; Tomczak, N.; Yang, Z.; Tang, B. Z.; Kong, D.; Liu, B. Precise and Long-Term Tracking of Adipose-Derived Stem Cells and Their Regenerative Capacity via Superb Bright and Stable Organic Nanodots. ACS Nano 2014, 8, 12620−12631. (24) Ding, D.; Goh, C. C.; Feng, G.; Zhao, Z.; Liu, J.; Liu, R.; Tomczak, N.; Geng, J.; Tang, B. Z.; Ng, L. G. Ultrabright Organic Dots with Aggregation-Induced Emission Characteristics for RealTime Two-Photon Intravital Vasculature Imaging. Adv. Mater. 2013, 25, 6083−6088. (25) Xu, S.; Yuan, Y.; Cai, X.; Zhang, C.-J.; Hu, F.; Liang, J.; Zhang, G.; Zhang, D.; Liu, B. Tuning the Singlet-Triplet Energy Gap: a Unique Approach to Efficient Photosensitizers with AggregationInduced Emission (AIE) Characteristics. Chem. Sci. 2015, 6, 5824− 5830. (26) Yuan, Y.; Zhang, C. J.; Kwok, R. T.; Xu, S.; Zhang, R.; Wu, J.; Tang, B. Z.; Liu, B. Light-Up Probe for Targeted and Activatable Photodynamic Therapy with Real-Time in Situ Reporting of Sensitizer Activation and Therapeutic Responses. Adv. Funct. Mater. 2015, 25, 6586−6595. (27) Zhang, R.; Feng, G.; Zhang, C. J.; Cai, X.; Cheng, X.; Liu, B. Real-Time Specific Light-Up Sensing of Transferrin Receptor: ImageGuided Photodynamic Ablation of Cancer Cells through Controlled Cytomembrane Disintegration. Anal. Chem. 2016, 88, 4841−4848. (28) Yuan, Y.; Feng, G.; Qin, W.; Tang, B. Z.; Liu, B. Targeted and Image-Guided Photodynamic Cancer Therapy Based on Organic Nanoparticles with Aggregation-Induced Emission Characteristics. Chem. Commun. 2014, 50, 8757−8760. (29) Hu, F.; Huang, Y.; Zhang, G.; Zhao, R.; Yang, H.; Zhang, D. Targeted Bioimaging and Photodynamic Therapy of Cancer Cells with an Activatable Red Fluorescent Bioprobe. Anal. Chem. 2014, 86, 7987−7995. (30) Yuan, Y.; Xu, S.; Zhang, C.-J.; Liu, B. Light-Responsive AIE Nanoparticles with Cytosolic Drug Release to Overcome Drug Resistance in Cancer Cells. Polym. Chem. 2016, 7, 3530−3539. (31) Yuan, Y.; Zhang, C. J.; Liu, B. A Photoactivatable AIE Polymer for Light-Controlled Gene Delivery: Concurrent Endo/Lysosomal Escape and DNA Unpacking. Angew. Chem., Int. Ed. 2015, 54, 11419− 11423. (32) Qin, W.; Ding, D.; Liu, J.; Yuan, W. Z.; Hu, Y.; Liu, B.; Tang, B. Z. Biocompatible Nanoparticles with Aggregation-Induced Emission Characteristics as Far-Red/Near-Infrared Fluorescent Bioprobes for in Vitro and in Vivo Imaging Applications. Adv. Funct. Mater. 2012, 22, 771−779. (33) Kamkaew, A.; Lim, S. H.; Lee, H. B.; Kiew, L. V.; Chung, L. Y.; Burgess, K. BODIPY Dyes in Photodynamic Therapy. Chem. Soc. Rev. 2013, 42, 77−88. (34) Desgrosellier, J. S.; Cheresh, D. A. Integrins in Cancer: Biological Implications and Therapeutic Opportunities. Nat. Rev. Cancer 2010, 10, 9−22.

(35) Cai, Y.; Tang, Q.; Wu, X.; Si, W.; Zhang, Q.; Huang, W.; Dong, X. Bromo-Substituted Diketopyrrolopyrrole Derivative with Specific Targeting and High Efficiency for Photodynamic Therapy. ACS Appl. Mater. Interfaces 2016, 8, 10737−10742. (36) Yu, T.; Malugin, A.; Ghandehari, H. Impact of Silica Nanoparticle Design on Cellular Toxicity and Hemolytic Activity. ACS Nano 2011, 5, 5717−5728. (37) Mazzarino, L.; Loch-Neckel, G.; Dos Santos Bubniak, L.; Ourique, F.; Otsuka, I.; Halila, S.; Curi Pedrosa, R.; Santos-Silva, M. C.; Lemos-Senna, E.; Curti Muniz, E.; Borsali, R. Nanoparticles Made from Xyloglucan-Block-Polycaprolactone Copolymers: Safety Assessment for Drug Delivery. Toxicol. Sci. 2015, 147, 104−115. (38) Xiong, L.; Yang, T.; Yang, Y.; Xu, C.; Li, F. Long-Term in Vivo Biodistribution Imaging and Toxicity of Polyacrylic Acid-Coated Upconversion Nanophosphors. Biomaterials 2010, 31, 7078−7085. (39) Wang, S.; Huang, P.; Nie, L.; Xing, R.; Liu, D.; Wang, Z.; Lin, J.; Chen, S.; Niu, G.; Lu, G.; Chen, X. Single Continuous Wave Laser Induced Photodynamic/Plasmonic Photothermal Therapy Using Photosensitizer-Functionalized Gold Nanostars. Adv. Mater. 2013, 25, 3055−3061. (40) Voon, S. H.; Kiew, L. V.; Lee, H. B.; Lim, S. H.; Noordin, M. I.; Kamkaew, A.; Burgess, K.; Chung, L. Y. In Vivo Studies of Nanostructure-Based Photosensitizers for Photodynamic Cancer Therapy. Small 2014, 10, 4993−5013.

3932


One-Step Formulation of Targeted Aggregation ... - ACS Publications

Recommend Documents